Anti-cancer cyclopenta [g] quinazoline compounds

ABSTRACT

Cyclopenta[g]quinazolines of the formula (I):—wherein: A is a group OR or NR 0 R 1  wherein R 0  and R 1  are each independently hydrogen C 1-4  alkyl, C 3-4  alkenyl, C 3-4  alkynyl, C 2-4  hydroxyalkyl, C 2-4  halogenoalkyl or C 1-4  cyanoalkyl, or R 0  and R 1  together with the intermediate N form a five- or six-membered heterocyclic ring; p is an integer in the range 1 to 4; R 2  is hydrogen, C 1-4  alkyl, C 3-4  alkenyl, C 3-4  alkynyl, C 2-4  hydroxyalkyl, C 2-4  halogenoalkyl or C 1-4  cyanoalkyl; Ar 1  is phenylene, thiophenediyl, thiazolediyl, pyridinediyl or pyrimidinediyl which may optionally bear one or two substituents selected from halogeno, hydroxy, amino, nitro, cyano, trifluoromethyl, C 1-4  alkyl and C 1-4  alkoxy; and R 3  is a group of one of the following formulae: -A 1 —Ar 2 -A 2 -Y 1 -A 5 -CON(R)CH(Y 4 )Y 5 -A 8 -X—Ar 4  and pharmaceutically acceptable salts or esters thereof are of therapeutic value particularly in the treatment of cancer.

This application is a continuation of application Ser. No. 11/710,985,filed Feb. 27, 2007, U.S. Pat. No. 7,863,284, which is a division ofapplication Ser. No. 10/487,871, filed Jul. 16, 2004, U.S. Pat. No.7,297,701, which is a 371 of PCT/GB02/03979, filed Aug. 30, 2002, whichclaims the benefit of priority to British Application No. 0121151.5,filed Aug. 31, 2001, Application Ser. No. 60/340,253, filed Dec. 18,2001, and British Application No. 0129387.7, filed Aug. 30, 2002, theentire content of which is hereby incorporated by reference in thisapplication.

This invention relates to novel anti-cancer agents and more particularlyit relates to cyclopenta[g]quinazoline derivatives which possessantiproliferative activity.

One group of anti-cancer agents comprises antimetabolites havingantifolate activity, such as the dihydrofolate reductase inhibitor,methotrexate and the thymidylate synthase (TS) inhibitors CB3717,raltitrexed and ZD9331. CB3717 is described and claimed in EP-B-0031237,raltitrexed in EP-B-0239362 and ZD9331 in EP-B-0562734. All of these TSinhibitors have demonstrable clinical activity in a range of solidtumours (see Cancer Treatment Reports, 1986, 70, 1335 and Beale et al.,“Tomudex: Clinical Development” in Antifolate Drugs in Cancer Therapy(ed. Jackman), Humana Press, Totowa, N.J., USA, pp. 177-181, 1999).Side-effects of raltitrexed and ZD9331 are predominantly related toinhibition of TS in gut and bone-marrow.

TS catalyses the methylation of deoxyuridine monophosphate to producethymidine monophosphate which is required for DNA synthesis. Theanticancer activity of these agents may be assessed in vitro bydetermining their inhibitory effect on that enzyme, and in cell culturesby their inhibitory effect on a range of mouse and human cancer celllines (see Boyle et al., “ZD9331: Preclinical and clinical studies” inAntifolate Drugs in Cancer Therapy (ed. Jackman), Humana Press, Totowa,N.J., USA, pp. 243-260, 1999 and Hughes et al., “Raltitrexed (Tomudex),a highly polyglutamatable antifolate thymidylate synthase inhibitor:design and preclinical activity” in Antifolate Drugs in Cancer Therapy(ed. Jackman), Humana Press, Totowa, N.J., USA, pp. 147-165, 1999).

More recently, cyclopenta[g]quinazoline derivatives showing a good levelof activity both as regards their ability to inhibit TS and also asregards their anticancer activity against various cell lines have beendeveloped.

WO-A-94/11354 (British Technology Group Limited) discloses tricycliccompound of formula:

wherein R¹ is hydrogen, amino, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄hydroxyalkyl or C₁₋₄ fluoroalkyl;

R² is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄hydroxyalkyl C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl;

Ar is phenylene, thiophenediyl, thiazolediyl, pyridinediyl orpyrimidinediyl which may optionally bear one or two substituentsselected from halogeno, hydroxy, amino, nitro, cyano, trifluoromethyl,C₁₋₄ alkyl and C₁₋₄ alkoxy; and

R³ is a group of one of the following formulae:—NHCH(CO₂H)-A¹-Y¹—NH-A³-Y³

or R³ is a N-linked naturally-occurring amino acid selected from thegroup consisting of L-alanine, L-leucine, L-isoleucine, L-valine andL-phenylalanine. Among the compounds disclosed is the L-Glu-γ-D-Glucompound CB300638, also mentioned in Clinical Cancer Research, 5,November 1999 (Supplement) at #566 (Theti et al.) and Proceedings of theAmerican Association for Cancer Research, 41, March 2000 at #33 (Jackmanet al.), as well as in J. Med. Chem., 2000, 43, 1910-1926, where it isdisclosed on page 1923 as compound 7b.

WO-A-95/30673 (British Technology Group Limited) disclosescyclopenta[g]quinazolines of formula:

wherein R¹ is hydrogen, amino, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄hydroxyalkyl or C₁₋₄ fluoroalkyl;

R² is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄hydroxyalkyl, C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl;

Ar¹ is phenylene, thiophenediyl, thiazolediyl, pyridinediyl orpyrimidinediyl which may optionally bear one or two substituentsselected from halogeno, hydroxy, amino, nitro, cyano, trifluoromethyl,C₁₋₄ alkyl and C₁₋₄ alkoxy; and

R³ is a group of one of the following formulae:-A¹-Ar²-A²-Y¹-A⁵-CON(R)CH(Y⁴)Y⁵-A⁸-X—Ar⁴

The α-isoform of the folate receptor (α-FR; membrane-associatedfolate-binding protein) is a glycosylphosphatidylinositol anchored cellmembrane protein that has very high affinity for folic acid and the morebiologically relevant reduced-folates (Kd ˜0.1 nM). The mechanism offolate internalisation is receptor-mediated endocytosis. The α-FR isoverexpressed in many carcinomas, particularly those of ovarian originwhere it is overexpressed highly and homogeneously in 90% of cases; seeCancer Res. 51, 5329-5338, 1991 (Campbell et al., 1991). Furthermore,high α-FR expression has been linked to aggressive, platinum resistantdisease and poor prognosis—see Int. J. Cancer 74, 193-198, 1997 and Int.J. Cancer 79, 121-126, 1998 (both Toffoli et al.). The β-isoform iswidely expressed in tumours of epithelial and non-epithelial origin withexpression levels being generally low/moderate and high, respectively,reviewed in Critical Rev. Therap. in Drug Carrier Systems 15, 587-627,1998 (Reddy and Low).

Folate receptors (α and β) are expressed in some adult normal tissues(low to moderate expression). Significant expression of the α-FR islargely restricted to kidney proximal tubules and choroid plexusalthough it is suggested that it is localised to the apical membranesurface in these organs and therefore may not play a significant role infolate uptake from blood (Reddy and Low, ibid.). There may be aspecialised function of the α-FR in the proximal tubules of the kidneyto salvage folates that escape in the filtrate.

The α-FR is hypothesised to be involved in cell signalling pathways. Forexample, in IGROV-1 ovarian carcinoma cells, immunoprecipitationexperiments have shown that the α-FR is associated in membranes with theG protein G_(α1-3), and the non-receptor kinase lyn.

High FR expression in some tumours relative to normal tissues is beingexploited in several areas of cancer medicine, including the selectivetumour delivery of conjugates of folic acid and toxins, liposomes,imaging or cytotoxic agents (Reddy and Low, ibid.). For example, folicacid-deferroxamine-¹¹¹In conjugates are detected only in FR-expressingtumours and not normal tissues of mice, with the exception of kidneyepithelial cells. The high selectivity of this approach resides in thevery low and high affinities of folic acid (not a major component ofplasma) for the RFC (reduced-folate carrier) and FR respectively. Thusantifolate drugs with similarly low and high affinity for the RFC andα-FR respectively could be highly selective for α-FR over-expressingtumours relative to normal tissues. In contrast with the folic acidconjugates they would not require intracellular cleavage to be active.

We have now discovered that certain compounds within the general classof cyclopenta[g]quinazolines have an unexpectedly high level ofselectivity for α-folate receptor expressing human tumour cell lines.Accordingly the present invention comprises a cyclopenta[g]quinazolineof formula (I):

wherein:

A is a group OR⁰ or NR⁰R¹ wherein R⁰ and R¹ are each independentlyhydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄ hydroxyalkyl,C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl, or R⁰ and R¹ together with theintermediate N form a five- or six-membered heterocyclic ring;

p is an integer in the range 1 to 4;

R² is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl, C₃₋₄ alkynyl, C₂₋₄hydroxyalkyl, C₂₋₄ halogenoalkyl or C₁₋₄ cyanoalkyl;

Ar¹ is phenylene, thiophenediyl, thiazolediyl, pyridinediyl orpyrimidinediyl which may optionally bear one or two substituentsselected from halogeno, hydroxy, amino, nitro, cyano, trifluoromethyl,C₁₋₄ alkyl and C₁₋₄ alkoxy; and

R³ is a group of the formula:-A¹-Ar²-A²-Y¹

in which A¹ is a bond between the α-carbon atom of the group—CO—NH—CH(CO₂H)— and Ar² or is a C₁₋₂ alkylene group;

Ar² is phenylene, tetrazolediyl, thiophenediyl, thiazolediyl,pyridinediyl or pyrimidinediyl which in the case of phenylene mayoptionally bear one or two substituents on the ring selected fromhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy;

A² is a C₁₋₃ alkylene or C₂₋₃ alkenylene group; and

Y¹ is carboxyl, tetrazol-5-yl, N—(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenyl-sulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; or

Y¹ is a group of the formula:—CON(R)CH(Y²)Y³

in which R is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl or C₃₋₄ alkynyl;

Y² is carboxy, tetrazol-5-yl, N—(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenyl-sulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; and

Y³ is the residue of a naturally occurring amino acid NH₂CH(CO₂H)Y³; or

Y³ is a group of the formula:-A⁴-CO₂H

in which A⁴ is a C₂₋₆ alkylene group;

R³ is a group of the formula:-A⁵-CON(R)CH(Y⁴)Y⁵

in which A⁵ is a C₁₋₆ alkylene group and R is as defined above;

Y⁴ is carboxy, tetrazol-5-yl, N—(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenyl-sulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; and

Y⁵ is the residue of a naturally occurring amino acid NH₂CH(CO₂H)Y⁵; or

Y⁵ is a group of the formula:-A⁴-CO₂H

in which A⁴ is as defined above; or

Y⁵ is a group of the formula:-A⁶-Ar³-A⁷-Y⁶

in which A⁶ is a bond between the α-carbon atom of the group-A⁵-CON(R)CH(Y⁴)— and Ar³ or is a C₁₋₂ alkylene group;

Ar³ is phenylene, tetrazolediyl, thiophenediyl, thiazolediyl,pyridinediyl or pyrimidinediyl which in the case of phenylene mayoptionally bear one or two substituents on the ring selected fromhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy;

A⁷ is a C₁₋₃ alkylene or C₂₋₃ alkenylene group; and

Y⁶ is carboxy, tetrazol-5-yl, N—(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenyl-sulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; or

R³ is a group of the formula:-A⁸-X—Ar⁴

in which A⁸ is a C₁₋₄ alkylene group;

X is sulfinyl, sulfonyl or methylene; and

Ar⁴ is 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl or, except when X ismethylene, tetrazol-5-yl;

the compound (I) optionally being in the form of a pharmaceuticallyacceptable salt or ester.

In this specification the terms alkyl, alkenyl, alkynyl and alkyleneinclude both straight and branched chain groups but references toindividual alkyl or alkylene groups, such as “propyl”, are specific forthe straight chain group only. An analogous convention applies to othergeneric terms. Moreover, the numbering system used for thecyclopenta[g]quinazoline nucleus is the conventional one as shown below:

Amino-acid residues are designated herein in the standard manner (Pureand Applied Chemistry, 1974, 40, 317 and European Journal ofBiochemistry, 1984, 138, 9). Thus, for example, γ-glutamyl denotes theradical H₂NCH(CO₂H)CH₂CH₂CO— or —NHCH(CO₂H)CH₂CH₂CO— according to thecontext, the carbon atoms in these radicals being numbered from thecarbon atom of the α-carboxy group as position 1.

It will be observed that a cyclopenta[g]quinazoline of the inventioncontains at least two asymmetric carbon atoms [present at the point ofattachment of the group —N(R²)— to the tricyclic ring system and at theα-carbon atom of the group —CONHCH(CO₂H)—] and can therefore exist inracemic and optically active forms. It is to be understood that thisinvention encompasses both racemic and optically active forms of thephysiologically active cyclopenta[g]quinazolines, it being a matter ofcommon general knowledge how such optically active forms may be obtainedby stereospecific synthesis or by separation of a mixture of isomericcompounds. It will be appreciated that one isomer may be of moreinterest than another due to the nature of the activity which itexhibits or due to superior physical properties, for example aqueoussolubility.

It is also to be understood that a cyclopenta[g]quinazoline of theformula (I) may exhibit the phenomenon of tautomerism and that theformulae shown in this specification represent only one of the possibletautomeric forms. Moreover, it will be appreciated that when, forexample, Y¹, Y², Y⁴ or Y⁶ is a tetrazol-5-yl group, that group may be inthe form of a 1H-tetrazol-5-yl group or a 2H-tetrazol-5-yl group. It isto be understood therefore that the invention is not limited merely toany one tautomeric form which is illustrated.

It is also to be understood that certain cyclopenta[g]quinazolines ofthe formula (I) can exist in solvated as well as unsolvated forms suchas, for example, hydrated forms.

A suitable value for R⁰, R¹ or R² when it is C₁₋₄ alkyl, or for a C₁₋₄alkyl substituent which may be present on Ar¹, Ar² or Ar³ or on a phenylgroup-containing group Y¹, Y², Y⁴ or Y⁶ present in R³, or for a group Rpresent in R³ when it is C₁₋₄ alkyl, is, for example, methyl, ethyl,propyl or isopropyl.

A suitable value for a C₁₋₄ alkoxy substituent which may be present onAr¹, Ar² or Ar³ or on a phenyl-containing group Y¹, Y², Y⁴ or Y⁶ is, forexample, methoxy, ethoxy, propoxy, isopropoxy or butoxy.

A suitable value for a halogeno substituent which may be present on Ar¹,Ar² or Ar³ or on a phenyl-containing group Y¹, Y², Y⁴ or Y⁶ is, forexample, fluoro, chloro or bromo.

A suitable value for R⁰, R¹ and R² when it is C₃₋₄ alkenyl or for agroup R present in R³ when it is alkenyl, is, for example, prop-2-enyl,but-2-enyl, but-3-enyl or 2-methylprop-2-enyl; and when it is C₃₋₄alkynyl is, for example, prop-2-ynyl or but-3-ynyl.

A suitable value for R⁰, R¹ and R² when it is C₂₋₄ hydroxyalkyl is, forexample, 2-hydroxyethyl or 3-hydroxypropyl; when it is C₂₋₄halogenoalkyl is, for example, 2-fluoroethyl, 2-chloroethyl,2-bromoethyl, 3-fluoropropyl, 3-chloropropyl or 3-bromopropyl; and whenit is C₁₋₄ cyanoalkyl is, for example, cyanomethyl, 2-cyanoethyl or3-cyanopropyl.

When R⁰ and R¹ together with the intermediate N form a five- orsix-membered heterocyclic ring, this may bear substituents, but the ringis preferably an unsubstituted saturated ring such as pyrrolidine orpiperidine.

A suitable value for Ar¹, Ar² or Ar³ when it is phenylene is, forexample, 1,3- or 1,4-phenylene, especially 1,4-phenylene.

A suitable value for Ar¹, Ar² or Ar³ when it is thiophenediyl is, forexample, thiophene-2,4-diyl or thiophene-2,5-diyl; when it isthiazolediyl is, for example thiazole-2,4-diyl or thiazole-2,5-diyl;when it is pyridinediyl is, for example, pyridine-2,4-diyl,pyridine-2,5-diyl, pyridine-2,6-diyl or pyridine-3,5-diyl; and when itis pyrimidinediyl is, for example, pyrimidine-2,4-diyl,pyrimidine-2,5-diyl or pyrimidine-4,6-diyl.

As indicated, Ar¹ and a phenylene group Ar² or Ar³ or a phenyl group inY¹ may carry one or two substituents. A preferred level of substitutionin Ar¹, where substitution is present, is either two substituents orespecially one substituent; and the one or two substituents mayconveniently be at positions adjacent to the atom bonded to the group—CONHCH(CO₂H)—R³, halogeno substituents such as fluoro being preferred.A preferred level of substitution on a phenylene group Ar² or Ar³ or ona phenyl group in Y¹, where substitution is present, is one substituent.

When R³ is a group of the formula:-A¹-Ar²-A²-Y¹

a suitable value for A¹ when it is a C₁₋₂ alkylene group is, for examplemethylene or ethylene and for A² when it is a C₁₋₃ alkylene group is,for example, methylene, ethylene or trimethylene. A suitable value forA² when it is a C₂₋₃ alkenylene group is, for example, vinylene orespecially propenylene (—CH₂CH═CH— or —CH═CH—CH₂—). A preferred valuefor both A¹, when it is not a bond, and for A² is methylene or ethylene.Suitable values for Ar² include those which have been discussedhereinbefore, such as thiophenediyl or most especially phenylene, oradditionally tetrazole-1,5-diyl or tetrazole-2,5-diyl. A suitable valuefor Y¹ or for Y² in a group Y¹ of formula —CON(R)CH(Y²)Y³ when it isN—(C₁₋₄ alkylsulfonyl)carbamoyl is, for example,N-methylsulfonylcarbamoyl, N-ethyl-sulfonylcarbamoyl orN-propylsulfonylcarbamoyl.

In a group Y¹ of formula —CON(R)CH(Y²)Y³ suitable values for C₁₋₄ alkyl,C₃₋₄ alkenyl and C₃₋₄ alkynyl groups R have been discussed hereinbeforebut R is preferably either methyl or especially hydrogen; a suitablevalue for Y³ when it is the residue of a naturally occurring amino acidis the residue of alanine (Y³═CH₃), arginine (Y³═(CH₂)₃NHC(NH₂)═NH),aspartic acid (Y³═CH₂CO₂H), cysteine (Y³═CH₂SH), isoleucine(Y³═CH(CH₃)CH₂CH₃), leucine (Y³═CH₂CH(CH₃)CH₃), ornithine(Y³═(CH₂)₃NH₂), phenylalanine (Y³═CH₂C₆H₅), serine (Y³═CH₂OH) and valine(Y³═CH(CH₃)₂) and especially glutamic acid (Y³═CH₂CH₂CO₂H). A suitablevalue for A⁴ when Y³ is a group of the formula -A⁴-CO₂H is trimethylene,pentamethylene or hexamethylene, A⁴ preferably being a C₃₋₆ alkylenegroup with especially suitable values for Y³ being —(CH₂)_(n)CO₂H wheren is 3, 4 or 5.

A preferred value for Y¹ or for Y², Y⁴ and Y⁶, is tetrazol-5-yl orespecially carboxy.

When R³ is a group of the formula:-A⁵-CON(R)CH(Y⁴)Y⁵

a suitable value for A⁵ is, for example, methylene, ethylene,trimethylene, tetramethylene, pentamethylene or hexamethylene and asuitable value for R is as described hereinbefore. A suitable value forY⁴ when it is N—(C₁₋₄ alkylsulfonyl)-carbamoyl is, for example,N-methylsulfonylcarbamoyl, N-ethylsulfonylcarbamoyl orN-propylsulfonylcarbamoyl.

A suitable value for Y⁵ when it is the residue of a naturally occurringamino acid is alanine (Y⁵=is CH₃), arginine (Y⁵=is (CH₂)₃NHC(NH₂)═NH),aspartic acid (Y⁵=is CH₂CO₂H), cysteine (Y⁵=is CH₂SH), isoleucine (Y⁵=isCH(CH₃)CH₂CH₃), leucine (Y⁵=is CH₂CH(CH₃)CH₃), ornithine (Y⁵=is(CH₂)₃NH₂), phenylalanine (Y⁵=is CH₂C₆H₅), serine (Y⁵=is CH₂OH), valine(Y⁵=is CH(CH₃)₂), and especially glutamic acid (Y⁵=is CH₂CH₂CO₂H). WhenY⁵ is a group of the formula -A⁴-CO₂H, suitable values for A⁴ and Y⁵ areas described hereinbefore in relation to a group Y³ of the formula-A4-CO₂H.

A suitable value for A⁶ in a group Y⁵ of the formula -A⁶-Ar³-A⁷-Y⁶ is asdescribed hereinbefore for A¹ and for A⁷ is as described for A². Asuitable value for Ar³ is as described hereinbefore for Ar². A suitablevalue for Y⁶ in such a group Y⁵ when it is N—(C₁₋₄alkylsulfonyl)carbamoyl is, for example, N-methylsulfonyl-carbamoyl,N-ethylsulfonylcarbamoyl or N-propylsulfonylcarbamoyl.

When R³ is a group of the formula:-A⁸-X—Ar⁴

a suitable value for A⁸ is, for example, methylene, ethylene,trimethylene or tetramethylene.

Groups R³ of particular value have the formula -A¹-Ar²-A²-Y¹, especiallywhen Y¹ is a group not of the formula —CON(R)CH(Y²)Y³, such as carboxyor tetrazol-5-yl.

Specific examples of such groups R³ are groups -A¹-Ar²-A²-Y¹ in which A¹is a bond or methylene or ethylene, Ar² is phenylene, thiophenediyl ortetrazolediyl, A² is methylene, ethylene or trimethylene and Y¹ iscarboxy or tetrazol-5-yl.

Other groups R³ of particular value have the formula -A⁵-CON(R)CH(Y⁴)Y⁵,especially when Y⁵ is a group of the formula -A⁶-Ar³-A⁷-Y⁶ in which Ar³is phenylene, thiophenediyl or tetrazolediyl.

Specific examples of such groups R³ are groups-A⁵-CON(R)CH(Y⁴)-A⁶-Ar³-A⁷-Y⁶ in which A⁵ is methylene or ethylene, R ishydrogen or methyl, Y⁴ is carboxy or tetrazol-5-yl, A⁶ is a bond or ismethylene or ethylene, Ar³ is phenylene, thiophenediyl or tetrazolediyl,A⁷ is methylene, ethylene or trimethylene and Y⁶ is carboxy ortetrazol-5-yl.

A suitable pharmaceutically-acceptable salt form of acyclopenta[g]quinazoline of the invention is, for example, an acidaddition salt with an inorganic or organic acid, for examplehydrochloric, hydrobromic, trifluoroacetic or maleic acid; or an alkalimetal, for example sodium, an alkaline earth metal, for example calcium,or ammonium, for example tetra(2-hydroxyethyl)ammonium, salt.

A suitable pharmaceutically-acceptable ester form of acyclopenta[g]quinazoline of the invention is, for example, an ester withan aliphatic alcohol of up to 6 carbon atoms, for example a methyl,ethyl or tert-butyl ester.

It is to be understood that R³ may contain several carboxy groups inaddition to the carboxy group in the grouping —CONHCH(CO₂H)—. When, forexample, two carboxy groups are present in the cyclopenta[g]quinazoline,a salt or ester may be mono-acid-mono-salt or -ester, di-salt ordi-ester and when, for example, three carboxy groups are present a saltor ester may be mono-acid-di-salt or -ester, di-acid-mono-salt or -esteror even tri-salt or -ester.

Particularly preferred values for the various symbols R⁰, R¹, R² and Ar¹individually are as expressed for the preferredcyclopenta[g]quinazolines described hereinafter.

A preferred cyclopenta[g]quinazoline of the invention has the formula(I) wherein R⁰ and R¹ are each independently hydrogen or C₁₋₄ alkyl,especially methyl;

wherein R² is ethyl, propyl, prop-2-enyl, prop-2-ynyl, 2-hydroxyethyl,2-fluoroethyl, 2-bromoethyl or 2-cyanoethyl;

wherein Ar¹ is 1,4-phenylene which may optionally bear one or twosubstituents selected from the group consisting of chloro and especiallyfluoro, thiophene-2,5-diyl, thiazole-2,5-diyl or pyridine-2,5-diyl;

wherein R³ is a group of the formula -A¹-Ar²-A²-Y¹ in which A¹ is a bondor is methylene or ethylene, Ar² is phenylene, A² is methylene, ethyleneor trimethylene and Y¹ is a group of the formula:—CON(R)CH(Y²)Y³

in which R is hydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl or C₃₋₄ alkynyl;

Y² is carboxy, tetrazol-5-yl, N—(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenyl-sulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; and

Y³ is the residue of a naturally occurring amino acid NH₂CH(CO₂H)Y³.Alternatively, a preferred cyclopenta[g]quinazoline of the invention hasthe formula (I) wherein:

R⁰ and R¹ are each independently hydrogen or C₁₋₄ alkyl;

R² is ethyl, propyl, prop-2-enyl, prop-2-ynyl, 2-hydroxyethyl,2-fluoroethyl, 2-bromoethyl or 2-cyanoethyl;

Ar¹ is 1,4-phenylene which may optionally bear one or two substituentsselected from the group consisting of chloro, fluoro,thiophene-2,5-diyl, thiazole-2,5-diyl or pyridine-2,5-diyl;

R³ is a group of the formula:-A⁵-CON(R)CH(Y⁴)Y⁵

in which A⁵ is a C₁₋₆ alkylene group and R is as defined above;

Y⁴ is carboxy, tetrazol-5-yl, N—(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenyl-sulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; and

Y⁵ is the residue of a naturally occurring amino acid NH₂CH(CO₂H)Y⁵.

A preferred value for p is 1.

A further preferred cyclopenta[g]quinazoline of the invention has theformula (I) wherein A is a group OR⁰ in which R⁰ is hydrogen or methyl;

wherein R² is ethyl or prop-2-ynyl; and

wherein Ar¹ is 1,4-phenylene or 1,4-phenylene having a 2-fluorosubstituent as in 2,6-difluoro-1,4-phenylene or especially2-fluoro-1,4-phenylene or is pyridine 2,5-diyl; and

R³ is as just described above.

An especially preferred cyclopenta[g]quinazoline of the invention hasthe formula (I) wherein A is a group OR⁰ in which R⁰ is hydrogen ormethyl;

wherein R² is ethyl or preferably prop-2-ynyl;

wherein Ar¹ is 1,4-phenylene or 2-fluoro-1,4-phenylene; and

wherein R³ is the residue of an acid NH₂.CH(COOH)R³ which comprisesL-Glu-γ-D-Glu, i.e. N-L-γ-glutamyl-D-glutamic acid.

Other quinazolines of the invention of particular interest have thevalues of R⁰, R¹, R², and Ar¹ and Ar in combination as indicated abovebut with R³ having any value as indicated hereinbefore. However,specific particularly preferred cyclopenta[g]quinazolines of theinvention are:

N-{N-{4-[N-((6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid;

N-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid; and

N-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamicacid;

or a pharmaceutically acceptable salt or ester thereof.

Although the compounds of the present invention can exist as a mixtureof stereoisomers it is preferred that they are resolved into oneoptically active isomeric form. Such a requirement complicates thesynthesis of the compounds and it is preferred therefore that theycontain as few asymmetric carbon atoms as possible consistent withachieving the desired activity.

As indicated previously, however, the cyclopenta[g]quinazolines of thepresent invention contain at least two asymmetric carbon atoms. Ofthese, that at the 6 position of the ring system preferably has the 6Sorientation rather than the 6R orientation, whilst the alpha carbon atomof the group —CONHCH(CO₂H)— preferably has the L rather than the Dconfiguration. The preferred compounds (I) described hereinbefore thuspreferably have such a configuration at these two asymmetric carbonatoms or less preferably are a racemic mixture in which one or both ofthese asymmetric carbon atoms is unresolved.

The asymmetric carbon atom of a residue R³ of the form-A¹-Ar²-A²-CON(R)CH(Y²)Y³ or -A⁵-CON(R)CH(Y⁴)Y⁵ may be of the L- orD-configuration but the amide bond will be stabilised in vivo when it isof the D-configuration as it will also be when R is other than hydrogen.When Y³ or Y⁵ is the residue of a naturally occurring amino acid,however, the amino acid intermediate for the synthesis of thecyclopenta[g]quinazoline will of course be more readily available whenthis asymmetric carbon atom is of the L-configuration.

A cyclopenta[g]quinazoline of the invention may be prepared by anyprocess known to be applicable to the preparation of chemically-relatedcompounds.

As stated above, cyclopenta[g]quinazolines of the present invention arebelieved to function as anti-cancer agents at least in part due to theirability to inhibit the enzyme thymidylate synthase. This anti-canceractivity may be assessed, for example, using one or more of theprocedures set out below:

(a) An in vitro assay which determines the ability of a test compound toinhibit the enzyme thymidylate synthase. Thymidylate synthase may beobtained in partially purified form from L1210 mouse leukaemia cells andutilised in the assay using the procedures described by Jackman et al.(Cancer Res., 1986, 46, 2810) and Sikora et al. (Biochem. Pharmacol.,1988, 37, 4047);

(b) An assay which determines the ability of a test compound to bind tothe α-FR relative to that of folic acid, using mouse L1210-FBP cells(α-FR expression) in the procedure described by Westerhof et al. (CancerRes., 1991, 51, 5507-5513);

(c) An assay which determines the ability of a test compound to inhibitthe growth of human tumour cell lines expressing the α-FR (A431-FBPvulvular carcinoma transfected with the α-FR; KB nasopharengealcarcinoma);

(d) An assay which determines the ability of a test compound to inhibitthe growth of human tumour cell lines not expressing the α-FR (A431neo-transfected);

(e) An assay confirming or demonstrating that compound-induced growthinhibition is largely attributable to α-FR mediated uptake into KB orA431-FBP cells. This involves the co-addition of an excess of folic acid(1 μM) to compete with the compounds for FR but not RFC binding.

Although the pharmacological properties of the cyclopenta[g]quinazolinesof the invention depend on their detailed structure, in general thecyclopenta[g]quinazolines of the invention possess activity in one ormore of the above tests (a) to (d) as indicated below:

-   -   Test (a) IC₅₀ in the range, for example, 0.0001-1 μM;    -   Test (b) Inverse relative affinity in the range, for example,        0.05-5 (values greater than 1 implies binding is greater than        that of folic acid and values less that 1 implies binding weaker        than that of folic acid);    -   Test (c) IC₅₀ in the range, for example, 0.001-10 μM;    -   Test (d) IC₅₀ in the range, for example, 0.01-100 μM;    -   Test (e) IC₅₀ at least 10-fold higher than that seen in test (b)        for the same cell line.

A cyclopenta[g]quinazoline of the present invention may itself be activeor it may be a pro-drug which is converted in vivo to an activecompound. A cyclopenta[g]quinazoline of the invention may beadministered to a warm-blooded animal, including a human, in the form ofa pharmaceutical composition which comprises thecyclopenta[g]quinazoline in association with apharmaceutically-acceptable diluent or carrier.

The composition may be in a form suitable for oral use, for example atablet, capsule, aqueous or oily solution, suspension or emulsion; aform suitable for topical use, for example a cream, ointment, gel oraqueous or oily solution or suspension; a form suitable for nasal use,for example a snuff, nasal spray or nasal drops; a form suitable forvaginal or rectal use, for example a suppository; a form suitable foradministration by inhalation, for example as a finely divided powdersuch as a dry powder, a microcrystalline form or a liquid aerosol; aform suitable for sub-lingual or buccal use, for example a tablet orcapsule; or a form suitable for parenteral use (including intravenous,subcutaneous, intramuscular, intravascular or infusion use), for examplea sterile aqueous or oily solution, emulsion or suspension. In generalthe above compositions may be prepared in a conventional manner usingconventional excipients.

The composition may contain, in addition to the cyclopenta[g]quinazolineof the invention, one or more other anti-cancer substances selectedfrom, for example, other antimetabolites, DNA interacting agents, signaltransduction inhibitors or other inhibitors of deregulated pathways intumours.

The cyclopenta[g]quinazoline will normally be administered to awarm-blooded animal at a dose within a range of 50-25000, particularly50-5000, mg per square metre body area of the animal, i.e. approximately1500, particularly 1-100, mg/kg. Where desired, however, dosages outsidethis range may be employed and, in particular, where the preferred modeof administration involving subcutaneous infusion is used then the doesrange may be increased to 1-1000 mg/kg. Preferably a daily dose in therange 10-250 mg/kg is employed, particularly 30-150 mg/kg. However, thedaily dose will necessarily be varied depending upon the host treated,the particular route of administration and the severity of the illnessbeing treated. Accordingly, the optimum dosage may be determined by thepractitioner who is treating any particular patient.

Accordingly the present invention also includes a method for aidingregression and palliation of cancer in a patient, particularly awarm-blooded animal such as a human, in need of such treatment, whichcomprises administering to said patient an effective amount of acyclopenta[g]quinazoline as defined hereinbefore. The invention alsoprovides the use of such a cyclopenta[g]quinazoline in the manufactureof a novel medicament for use in the treatment of cancer.

Cyclopenta[g]quinazolines of the present invention are of interest for awide range of anti-tumour activities particularly the treatment ofovarian cancer.

In view of the activity shown by antimetabolites such as aminopterin andmethotrexate, which is discussed hereinbefore, thecyclopenta[g]quinazolines of the present invention are also of interestfor use in the treatment of other conditions, for example allergicconditions such as psoriasis and inflammatory diseases such asrheumatoid arthritis. In using a cyclopenta[g]quinazoline of theinvention for such a purpose the compound will normally be administeredat a dose within the range 5-25000, particularly 5-500, mg per squaremetre body area of the animal, i.e. approximately 0.1-500, particularly0.1-10, mg/kg. Where desired, however, dosages outside this range may beemployed. In general, for the treatment of an allergic condition such aspsoriasis, topical administration of a cyclopenta[g]quinazoline of theinvention is preferred. Thus, for example, for topical administration adaily dose in the range, for example, of 0.1 to 10 mg/kg may be used.

Compositions containing the quinazolines may be formulated in unitdosage form, i.e. in the form of discrete portions each comprising aunit dose, or a multiple or sub-multiple of a unit dose, for example asa tablet or capsule. Such a unit dosage form may, for example, containan amount of the cyclopenta[g]quinazoline in the range of 1-250 or 1-500mg.

The invention is illustrated by the following Examples.

EXAMPLE 1 Synthesis of CB300951 (2-CH₂OMe Derivative of CB300638)

5-Methoxyacetamidoindan

To a solution of 5-aminoindan (4.66 g, 35.0 mmol) in anhydrous DMF (26ml) was slowly added methoxyacetyl chloride (5.70 g, 52.50 mmol)followed by pyridine (8.5 ml, 105.0 mmol). The red solution was stirredat room temperature for 3.5 hours under argon, then it was partitionedbetween ethyl acetate (200 ml) and 1N HCl (120 ml). The organic layerwas washed with more 1N HCl (120 ml), brine (100 ml), dried (Na₂SO₄) andconcentrated in vacuo. The residue was triturated with diethyl ether;the white precipitate was collected by filtration, washed with diethylether to afford the title compound (5.93 g, 83%), m.p. 104-105° C.;¹H-NMR (250 MHz, CDCl₃, TMS) 2.06 (m, 2H, 2-CH₂), 2.87 (m, 4H, 1-CH₂ and3-CH₂), 3.50 (s, 3H, OCH₃), 4.00 (s, 2H, 2-CH₂OMe), 7.22 (m (overlapwith CHCl₃ peak), 2H, 6-H, 7-H), 7.52 (s, 1H, 4-H), 8.18 (s, 1H, CONH);MS (ESI, m/z): 432 [(2M+Na)⁺, 30%], 206[(M+H)⁺, 100%]. Found C, 70.10;H, 7.38; N, 6.81; C₁₂H₁₅NO₂ requires C, 70.22; H, 7.37; N, 6.82%.

5-Methoxyacetamido-6-bromoindan

A mixture of 5-methoxyacetamidoindan (5.50 g, 0.027 mol) and glacialacetic acid (25 ml) was cooled in an ice-water bath (˜10° C.). Bromine(1.5 ml, 0.029 mol) was then dropwise added over a 20 min period whilethe temperature was kept between 10-15° C. The reaction mixture was thenstirred for a longer 1 hour and then it was poured into an ice-waterbath (100 ml) with the aid of water (70 ml). The precipitate wascollected by filtration, washed with plenty of water (150 ml) and driedin vacuo over P₂O₅ to afford the title compound (6.98 g, 91%), m.p.84-86° C.; ¹H-NMR (250 MHz, CDCl₃, TMS) 2.09 (m, 2H, 2-CH₂), 2.88 (m,4H, 1-CH₂ and 3-CH₂), 3.55 (s, 3H, OCH₃), 4.04 (s, 2H, 2-CH₂OMe), 7.34,8.22 (2×s, 2H, 4-H, 7-H), 8.83 (s, 1H, CONH);

MS (ESI, m/z): 284, 286 [(M+H)⁺, 98%, 100%; Br isotopic pattern]. FoundC, 50.62; H, 4.93; N, 4.92; Br, 28.05; C₁₂H₁₄BrNO₂ requires C, 50.72; H,4.97; N, 4.93; Br, 28.12%.

5-Methoxyacetamido-6-bromoindan-1-one

To a solution of 5-methoxyacetamido-6-bromoindan (0.85 g, 3.0 mmol) inglacial acetic acid (7 ml) heated at 55° C. was dropwise added asolution of CrO₃ (1.2 g, 12.0 mmol) in aqueous glacial acetic acid, (7ml; v/v 1:1) over a 15 min period. The reaction mixture was then stirredat this temperature for 45 min. The reaction mixture was cooled in anice-bath, then propan-2-ol (4 ml) was added and the mixture was stirredat this temperature for 10 min before being concentrated in vacuo. Theblack residue was broken up with a spatula with the aid of water andthen partitioned between water (50 ml) and ethyl acetate (150 ml). Theaqueous layer was extracted with more ethyl acetate (2×40 ml); thecombined extracts were dried (Na₂SO₄), and concentrated in vacuo to givean off white residue. Purification by column chromatography on elutionwith 5% ethyl acetate in dichloromethane afforded in order of elution:

a. 5-methoxyacetamido-6-bromoindan-1-one as a white solid which wasfurther purified by trituration with ethyl acetate/hexanes (1:5, v/v):0.50 g (55%), m.p. 162-163° C.; ¹H-NMR (250 MHz, CDCl₃, TMS) 2.72 (m,2H, 2-CH₂), 3.11 (m, 2H, 3-CH₂), 3.57 (s, 3H, OCH₃), 4.09 (s, 2H,2-CH₂OMe), 7.95 (s, 1H) and 8.65 (s, 1H) (2H, 4-H, 7-H), 9.27 (s, 1H,CONH); MS (ESI, m/z) 298, 300 {(M+H)⁺, 100%, 97% respectively, bromineisotopic pattern}. Found: C, 48.13; H, 3.99; N, 4.70; Br, 26.95;C₁₂H₁₂BrNO₃ requires C, 48.34; H, 4.06; N, 4.70; Br, 26.80%); and

b. 5-acetamido-6-bromoindan-3-one as a solid which was further purifiedby trituration with ethyl acetate/hexanes (1:5, v/v): 0.026 g, (3%),m.p. 149-151° C. ¹H-NMR (250 MHz, CDCl₃, TMS) 2.71 (m, 2H, 2-CH₂), 3.01(m, 2H, 1-CH₂), 3.56 (s, 3H, OCH₃), 4.08 (s, 2H, 2-CH₂OMe), 7.73 (s, 1H)and 8.71 (s, 1H) (2H, 4-H, 7-H), 8.97 (s, 1H, CONH); MS (ESI, m/z) 298,300 {(M+H)⁺, 100%, 98% respectively, bromine isotopic pattern}. Found:C, 47.95; H, 3.96; N, 4.59; Br, 26.63; C₁₂H₁₂BrNO₃ requires C, 48.34; H,4.06; N, 4.70; Br, 26.80%).

tert-Butyl 4-[N-(5-methoxyacetamido-6-bromoindan-1-yl)amino]benzoate

Method A: To a flask containing 5-methoxyacetamido-6-bromoindan-1-one(0.357 g, 1.2 mmol), 4-toluenesulfonic acid monohydrate (0.015 g), andtert-butyl 4-aminobenzoate (0.289 g, 1.5 mmol) was added1,2-dimethoxyethane (dried by distillation over CaH₂; 15 ml). An Aldrichazeotropic distillation apparatus containing molecular sieves (3A) wasfitted to the reaction flask that was placed in an oil bath preheated to115° C. The reaction mixture was stirred at this temperature for 3.5hours under argon; then allowed to cool to room temperature, and asolution of sodium cyanoborohydride in tetrahydrofuran (1M; 1.55 ml.1.55 mmol) was added followed immediately by acetic acid (0.044 ml). Theblack reaction mixture was stirred at room temperature for 1 hour underargon; then it was partitioned between ethyl acetate (150 ml) andsaturated aqueous sodium bicarbonate (100 ml). The aqueous layer wasextracted with more ethyl acetate (100 ml); the organic extracts werecombined, washed with brine (100 ml), dried (Na₂SO₄), and concentratedin vacuo to leave a reddish residue. Purification by columnchromatography, on elution with 35% ethyl acetate in petroleum ether(60-80° C.), afforded the desired product as a white solid: 0.175 g(31%).

Method B: To a nearly clear solution of5-methoxyacetamido-6-bromo-indan-1-one (0.300 g, 1.0 mmol) in anhydrousmethanol (40 ml) was added tert-butyl 4-aminobenzoate (0.193 g, 1.0mmol) followed by decaborane (0.044 g). The reaction mixture was stirredat room temperature for 24 hours before being concentrated in vacuo.Purification by column chromatography, on elution with 35% ethyl acetatein petroleum ether (60-80° C.), afforded a white solid that was furtherpurified by reprecipitation from dichloromethane/hexane: 0.340 g, (72%)m.p. 152-153° C.; ¹H-NMR (250 MHz, CDCl₃, TMS) 1.57 (s, 9H, C(CH₃)₃),1.93, 2.63 (2×m, 2H, indanyl 2-H), 2.97 (m, 2H, indanyl 3-H), 3.55 (s,3H, OCH₃), 4.06 (s, 2H, CH₂ OMe), 5.04 (t, J=6.50 Hz, 1H, 1-H), 6.64 (d,J=8.78 Hz, 2H, 3,5-H), 7.51, 8.33 (2×s, each 1H, indanyl 4-H, 7-H), 7.85(d, J=8.75 Hz, 2,6-H), 8.93 (s, 1H, CONH); MS (ESI, m/z) 499, 497{(M+Na)⁺, bromine isotopic pattern}.

tert-Butyl 4-[N-(5-methoxyacetamido-6-cyanoindan-1-yl)amino]benzoate

To a solution of tert-butyl4-[N-(5-methoxyacetamido-6-bromoindan-1-yl)-amino]benzoate (0.714 g,1.50 mmol) in NMP (8 ml) [1-methyl-2-pyrrolidone] was added copper (I)cyanide (0.230 g, 2.55 mmol). The reaction mixture was placed in anoil-bath preheated to 140° C. and stirred at this temperature for 2 h.More copper (I) cyanide (0.100 g, 1.10 mmol) was then added and stirringwas continued for a longer 3 hours. The reaction mixture was allowed tocool to room temperature, then poured into a mixture of aqueous ammonia(d=0.88, 7 ml) and ice (˜20 ml) and the resulting brown mixture wasstirred at room temperature for ˜5 min. The brown solid was collected byfiltration washed with plenty of water, then suspended indichloromethane (100 ml). The mixture was stirred at room temperaturefor 10 min, dried (Na₂SO₄), and concentrated in vacuo. Purification bycolumn chromatography, on elution with 35% ethyl acetate in hexane,afforded an off white solid that was reprecipitated fromdichloromethane-ethyl acetate/hexane: 0.328 g, (52%) m.p. 163-164° C.¹H-NMR (250 MHz, DMSO-d₆, TMS) 1.50 (s, 9H, C(CH₃)₃), 1.85, 2.58 (2×m,2H, indanyl 2-H), 2.89 (m, 2H, indanyl 3-H), 3.41 (s, 3H, OCH₃), 4.05(s, 2H, CH₂OMe), 5.06 (m, 1H, indanyl 1-H), 6.73 (d, J=8.82 Hz, 2H,3,5-H), 6.82 (d, J=8.37 Hz, 1H, N¹⁰—H), 7.59, 7.57 (2×s, each 1H,indanyl 4-H, 7-H), 7.66 (d, J=8.77 Hz, 2,6-H), 9.88 (s, 1H, CONH); MS(ESI, m/z) 444 {(M+Na)⁺, 100%}. Found: C, 68.21; H, 6.47; N, 9.81;C₂₄H₂₇N₃O₄ requires C, 68.39; H, 6.46; N, 9.97%.

tert-Butyl4-{N-[(6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]amino}benzoate

A mixture of tert-butyl4-[N-(5-methoxyacetamido-6-cyanoindan-1-yl)-amino]benzoate (0.295 g,0.70 mmol), ethanol (3.2 ml), and water (0.64 ml) was cooled in anice-bath, then 30% aqueous H₂O₂ solution (0.60 ml) was added followed bygranulated sodium hydroxide pellets (0.047 g, 1.19 mmol). The reactionmixture was stirred at ˜0° C. for 10 min, then it was placed in an oilbath preheated to 55° C. and stirred at this temperature for 30 min. Thereaction mixture was allowed to cool to room temperature, then thesolvents were removed in vacuo and the residue was suspended in water(˜15 ml). The pH of this mixture was adjusted to ˜12 with 1N NaOH (got aclear solution), then to ˜4 with 1N hydrochloric acid. The off whiteprecipitate was collected by filtration, washed with water, dried invacuo over P₂O₅: 0.262 g (89%), m.p.>122° C. (softens); ¹H-NMR (250 MHz,DMSO-d₆, TMS) 1.50 (s, 9H, C(CH₃)₃), 1.87, 2.56 (2×m, each 1H, 7-H),3.00 (m, 2H, 8-H), 3.34 (s, 3H, OCH₃), 4.30 (s, 2H, 2-CH₂), 5.16 (m, 1H,6-H), 6.78 (d, J=8.55 Hz, 2H, 3′,5′-H), 6.89 (d, J=8.10, N¹⁰—H), 7.52,7.90 (2×s, each 1H, 5-H, 9-H), 7.67 (d, J=8.45 Hz, 2′,6′-H), 12.12 (s,1H, N³—H); MS (ESI, m/z) 444 {(M+Na)⁺, 40%}. Found: C, 67.19; H, 6.31;N, 9.69; C₂₄H₂₇N₃O₄0.5H₂O requires C, 66.96; H, 6.50; N, 9.76%.

Dicobalthexacarbonyl propargyl Alcohol Complex

This is a known compound (K.-D. Roth and U. Muller, Tetrahedron Letters1993, 34, 2919) and in this study was prepared according to Nicholas'methodology (K. L. Salazar and K. M. Nicholas, Tetrahedron 2000, 56,2211): To a round bottom flask charged with Co₂(CO)₆ (5.12 g, 15.0 mmol)under argon in a well ventilated hood was added anhydrousdichloromethane (170 ml) followed by a solution of propargyl alcohol(0.840 g, 15.0 mmol) in anhydrous dichloromethane (20 ml). The deep redreaction mixture was stirred at room temperature for 7 h under argon,then it was filtered through a thin layer of neutral alumina. Thefiltrate was concentrated in vacuo to give a red residue. Purificationby column chromatography, on elution with 40% diethyl ether in hexane,afforded the desired product as a red solid 4.10 g (80%); ¹H-NMR (250MHz, CDCl₃, TMS) 1.83 (t, J=6.0 Hz, 1H, OH), 4.80 (d, J=6.0 Hz, 2H,CH₂), 6.08 (s, 1H, C—H).

(Propargyl)Co₂(CO)₆ ⁺BF₄ ⁻

This is a known compound (K.-D. Roth and U. Muller, Tetrahedron Letters1993, 34, 2919) and in this study was prepared according to Nicholas'methodology (K. L. Salazar and K. M. Nicholas, Tetrahedron 2000, 56,2211): To a round bottom flask charged with dicobalthexacarbonylpropargyl alcohol complex (1.60 g, 4.7 mmol) under argon was added(syringed via a septum) propionic acid (2.2 ml). The reaction mixturewas cooled to −20° C. and then a solution of HBF₄ in diethyl ether (54%w/w, 2.05 ml) was slowly syringed into the reaction mixture via aseptum. The reaction mixture was stirred at −20° C. for 40 min, thencooled diethyl ether (50 ml) was added. Trituration afforded a redprecipitate that was collected by filtration, washed with plenty of drydiethyl ether and dried in vacuo over P₂O₅: 1.71 g (90%). This wasimmediately used in the next reaction without any further purification.

tert-Butyl4-{N-[(6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoate

To a round-bottomed flask containing the tetrafluoroborate salt(propargyl)Co₂(CO)₆ ⁺BF₄ ⁻ (0.271 g, 0.66 mmol) was added anhydrousdichloro-methane (dried by distillation over P₂O₅; 22 ml). The nearlyclear red dark solution was stirred at room temperature for few minutesunder argon, then tert-butyl4-{N-[(6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]amino}benzoate(0.215 g, 0.51 mmol) was added in one portion. Stirring was continued atthis temperature for 5 min then diisopropylethylamine (0.18 ml, 1.04mmol) was added and the reaction mixture was stirred at room temperaturefor 45 min under argon. The reaction mixture was partitioned betweenethyl acetate (150 ml) and brine (60 ml). The aqueous layer wasextracted with more ethyl acetate (2×50 ml). The combined extracts werewashed with 10% aqueous citric acid (50 ml), brine (50 ml), dried(Na₂SO₄), and concentrated in vacuo. Purification by columnchromatography, on elution with 40% ethyl acetate in dichloromethane,gave a red solid: 0.285 g (75%). To a solution of this complex (0.267 g,0.36 mmol) in ethanol (60 ml) was added Fe(NO₃)₃.9H₂O (˜8.0 g). Theclear solution was stirred at room temperature for 10 min then a secondportion of Fe(NO₃)₃.9H₂O (˜4.0 g) was added. The reaction mixture wasstirred at room temperature for a longer 5 min then a final portion ofFe(NO₃)₃.9H₂O (˜5.0 g) was added; the nearly clear solution was turnedinto a dark red mixture. Stirring was continued at room temperature foran extra 25 min, then the reaction mixture was partitioned between ethylacetate (150 ml) and dilute brine (700 ml). The aqueous layer wasextracted with more ethyl acetate (2×70 ml), The combined organics werewashed with brine (3×70 ml), dried (Na₂SO₄), and concentrated in vacuo.Purification by column chromatography, on elution with 2% methanol indichloromethane, afforded a white solid which was reprecipitated fromdichloromethane/hexane: 0.122 g (74%), m.p. 191-192° C.; (250 MHz,DMSO-d₆, TMS) 1.51 (s, 9H, C(CH₃)₃), 2.23, 2.53 (m-obscured by DMSOpeak) (2×m, each 1H, 7-H), 2.95-3.20 (m, 3H, C≡CH, 8-H), 3.34 (s(obscured by the H₂O peak), 3H, OCH₃), 3.96 (ABq, J=18.0 Hz, 2H,CH₂C≡C), 4.31 (s, 2CH₂), 5.79 (t, J=7.0 Hz, 1H, 6-H), 7.02 (d, J=9.01Hz, 2H, 3′,5′-H), 7.58, 7.80 (2×s, each 1H, 5-H, 9-H), 7.76 (d, J=8.45Hz, 2′,6′-H), 12.17 (s, 1H, N³—H); MS (ESI, m/z) 482 {(M+Na)⁺, 10%}.Found: C, 70.32; H, 6.31; N, 9.09; C₂₇H₂₉N₃O₄ requires C, 70.57; H,6.36; N, 9.14%.

4-{N-[(6RS)-2-Methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoicacid

A solution of tert-butyl4-{N-[(6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetra-hydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoate(0.069 g, 0.15 mmol) in dichloromethane (1 ml) and trifluoroacetic acid(3 ml) was stirred at room temperature for 1 hour and 10 min, then thesolvents were removed in vacuo. The residue was triturated with diethylether and the precipitate was collected by filtration, washed withdiethyl ether and dried in vacuo over P₂O₅ to afford the title compoundas the trifluoroacetate salt: 0.061 g, m.p. 225° C. (dec); ¹H-NMR (250MHz, DMSO-d₆, TMS) 2.23, 2.53 (obscured by DMSO peak) (2×m, each 1H,7-H), 2.90-3.20 (m, 3H, C≡CH, 8-H), 3.34 (s (obscured by the H₂O peak),3H, OCH₃), 3.97 (ABq, J=18.0 Hz, 2H, CH₂C≡C), 4.32 (s, 2H, 2-CH₂), 5.79(t, J=8.1 Hz, 1H, 6-H), 7.03 (d, J=9.02 Hz, 2H, 3′,5′-H), 7.58, (s, 1H,9-H), 7.81 (m, 3H, 5-H, 2′,6′-H), 12.17 (s, 1H, N³—H); MS (ESI, m/z) 426{(M+Na)⁺, 25%}, 404 {(M+H)^(÷), 70%}.

Tri-tert-butylN-{N-{4-[N-((6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetra-hydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamate

To a mixture of4-{N-[(6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoicacid trifluoro-acetate salt (0.056 g, ˜0.14 mmol), tri-tert-butylL-γ-glutamyl-D-glutamate (0.090 g, 0.20 mmol) and anhydrous DMF (2.5 ml)was added diethyl cyanophosphonate (0.050 g, 0.31 mmol) followed bytriethylamine (0.035 g, 0.35 mmol). The clear solution was stirred atroom temperature for 2 hours, then it was partitioned between ethylacetate (150 ml) and water (80 ml). The aqueous layer was extracted withethyl acetate (2×70 ml). The combined organics were washed with 10%aqueous citric acid (2×40 ml), saturated sodium bicarbonate solution(100 ml) and brine (100 ml), dried (Na₂SO₄), and concentrated in vacuo.Purification by column chromatography, on elution with 1.5% methanol inethyl acetate, afforded a white solid that was further purified bytrituration with hexane with the aid of some dichloromethane: 0.072 g(64%); m.p.>120° C.; ¹H-NMR (250 MHz, DMSO-d₆, TMS) 1.38, 1.39, 1.41(3×s, 27H, 3×C(CH₃)₃), 1.60-2.35 (m, 9H, 2×β-CH₂, 2×γ-CH₂, 7-H), 2.52 (mobscured by DMSO peak, 1H, 7-H), 2.90-3.25 (m, 3H, C≡CH, 8-H), 3.97(ABq, J=17.0 Hz, 2H, CH₂C≡C), 4.08, 4.12 (2×m, 2H, 2×α-CH), 4.32 (s, 2H,2-CH₂), 5.77 (t, J=7.50 Hz, 1H, 6-H), 7.02 (d, J=8.85 Hz, 2H, 3′,5-H),7.58 (s, 1H, 9-H), 7.80 (d, J=9.0 z, 2H, 2′,6′-H), 7.82 (s, 1H, 5-H),8.17 (d, J=8.12 Hz, 1H, CONH), 8.36 (d, J=7.00 Hz, 1H, CONH), 12.16 (s,1H, N³—H); MS (ESI, m/z) 852 {(M+Na)⁺, 20%}, 830 {(M+H)⁺, 100%}. FoundC, 64.71; H, 7.21; N, 8.27. C₄₅H₅₉N₅O₁₀ requires C, 65.12; H, 7.17; N,8.44%.

N-{N-{4-[N-((6RS)-2-Methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid

A solution of tri-tert-butylN-{N-{4-[N-((6RS)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]-benzoyl}-L-γ-glutamyl}-D-glutamate(0.056 g, 0.07 mmol) in trifluoroacetic acid (4.5 ml) was stirred atroom temperature for 1 hour and 10 min with protection from the light.The solvent was then removed in vacuo and the residue was suspended inwater (4 ml). The pH was adjusted to ˜10 with 1N NaOH, then to ˜4 with1N hydrochloric acid. The white precipitate was collected by filtrationand dried in vacuo over P₂O₅: 0.020 g (45%), m.p. 150-155° C. (softens);¹H-NMR (250 MHz, DMSO-d₆, TMS) 1.60-2.30 (m, 9H, 2×β-CH₂, 2×γ-CH₂, 7-H),2.52 (m obscured by DMSO peak, 1H, 7-H), 2.90-3.25 (m, 3H, C≡CH, 8-H),3.36 (s, 3H, OCH₃), 3.97 (ABq, J=17.0 Hz, 2H, CH₂C≡C), 4.20, 4.34 (2×m,2H, 2×α-CH), 4.32 (s, 2H, 2-CH₂), 5.77 (t, J=8.02 Hz, 1H, 6-H), 7.02 (d,J=8.85 Hz, 2H, 3′,5-H), 7.58 (s, 1H, 9-H), 7.81 (d, J=9.0 z, 2H,2′,6′-H), 7.83 (s, 1H, 5-H), 8.14 (d, J=8.12 Hz, 1H, CONH), 8.33 (d,J=7.78 Hz, 1H, CONH), 12.10 (s, 1H, N³-H); MS (ESI, m/z) 662 {(M+H)⁺,100%}.

EXAMPLE 2 Synthesis of CB300945 (2-CH₂OH Derivative of CB300638)

2-Hydroxymethyl-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one

A solution of caesium acetate (14.4 g, 75.2 mmol) in dry DMF (40 ml) washeated to 60° C. under argon for 30 min. The mixture was cooled to 40°C. and a suspension of2-chloromethyl-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one (L.Skelton, V. Bavetsias, A. Jackman, WO 00/050417-A1; 2.2 g, 9.4 mmol) indry DMF (60 ml) was added via a cannula. The mixture was heated to 80°C. under argon for 16 h. The mixture was cooled to room temperature andthe solvent was removed in vacuo. The residue was suspended in water (50ml) and MeOH (20 ml). The pH was adjusted to 12.5 with 1M sodiumhydroxide solution and the brown suspension was stirred for 2 h at roomtemperature. The insoluble brown solid was removed by filtration and theresulting solution was acidified to pH 5 with 1M hydrochloric acid.

The precipitate was collected by filtration, washed with acidified waterand dried in vacuo over P₂O₅ to yield the product as a pale yellow solid(1.17 g, 58%); m.p. 205-210° C.; NMR (DMSO-d₆) δ 2.07 (quin, J=7.4 Hz,2H, 7-H), 2.98 (q, J=6.95 Hz, 4H, 6-H and 8-H), 4.38 (s, 2H, 2-CH₂),7.46 (s, 1H, 9-H), 7.92 (s, 1H, 5-H); MS (FAB-m/z). Found 217 [(M+H)⁺,100%]; HRMS: measured 217.0977; calculated for C₁₂H₁₃N₂O₂ (M+H)⁺:217.0977. Found C, 64.01; H, 5.23; N, 12.34. C₁₂H₁₃N₂O₂.½H₂O requires C,63.93; H, 5.77; N, 12.43%.

2-(2,2-Dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one

2-Hydroxymethyl-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one (1.0g, 4.6 mmol), triethylamine (0.77 ml, 5.6 mmol), DMAP (50 mg, 0.4 mmol)and anhydrous CH₂Cl₂ (50 ml) were mixed in a flask under argon. Pivalicanhydride (1.2 ml, 6.0 mmol) was added dropwise and the suspensionstirred at room temperature under argon for 5 h. The solvent was removedin vacuo and the residue partitioned between EtOAc (100 ml) andsaturated aqueous NaHCO₃ (100 ml). The organic extract was washed withsaturated aqueous NaHCO₃ (70 ml), water (70 ml), brine (70 ml), dried(Na₂SO₄) and the solvent removed in vacuo. The residue was trituratedwith hexane (60 ml) and the product collected by filtration as a yellowsolid (1.21 g, 87%); m.p. 185-190° C.; ¹H-NMR (DMSO-d₆) δ 1.22 (s, 9H,CMe₃), 2.07 (quin, J=7.4 Hz, 2H, 7-H), 2.98 (q, J=5.72 Hz, 4H, 6-H and8-H), 4.94 (s, 2H, 2-CH₂), 7.42 (s, 1H, 9-H), 7.92 (s, 1H, 5-H), 12.20(br, 1H, NH); MS (FAB, m/z). Found 301 [(M+H)⁺, 100%]; HRMS: measured301.1539; calculated for C₁₇H₂₁N₂O₃ (M+H)⁺: 301.1552. Found C, 67.65; H,6.54; N, 9.54. C₁₇H₂₀N₂O₃ requires C, 67.98; H, 6.71; N, 9.33%.

2-(2,2-Dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,6-dioneand2-(2,2-Dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,8-dione

To a stirred solution of (Ph₃SiO)₂CrO₂ (L. M. Baker and W. L. Carrick,J. Org. Chem. 1970, 35, 774) (10.6 mg, 0.017 mmol) in CH₂Cl₂ (5 ml) wasadded sequentially aqueous 70% tert-butyl hydroperoxide (0.18 ml, 1.3mmol) and2-(2,2-dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4-one(0.1 g, 0.33 mmol). The mixture was stirred at room temperature withprotection from the light for 24 h. The solvents were removed in vacuoand the residue purified by column chromatography (20 g of silica gel)eluting with a gradient of 10-30% EtOAc in CHCl₃ to yield2-(2,2-dimethylpropionyloxyrnethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,6-dioneas a white solid (47 mg, 45%); m.p. 185-190° C.; ¹H-NMR (DMSO-d₆) δ 1.23(s, 9H, CMe₃), 2.72 (m, 2H, 7-H), 3.25 (m, 2H, 8-H), 5.00 (s, 2H,2-CH₂), 7.70 (s, 1H, 9-H), 8.29 (s, 1H, 5-H), 12.20 (br, 1H, NH); MS(FAB, m/z). Found 315 [(M+H)⁺, 100%], 337 [(M+Na)⁺, 75%]; HRMS: measured315.1360; calculated for C₁₇H₁₉N₂O₄ (M+H)⁺: 315.1345. Found C, 64.18; H,5.72; N, 8.81. C₁₇H₁₈N₂O₄.0.2H₂O requires C, 64.23; H, 5.79; N, 8.82%.

2-(2,2-Dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,8-dione;¹H-NMR (DMSO-d₆) δ 1.23 (s, 9H, CMe₃), 2.76 (m, 2H, 7-H), 3.26 (m, 2H,8-H), 4.98 (s, 2H, 2-CH₂), 7.72 (s, 1H, 9-H), 8.29 (s, 1H, 5-H), 12.3(br, 1H, NH).

tert-Butyl4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)amino]benzoate

A suspension of2-(2,2-dimethylpropionyloxymethyl)-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-4,6-dione(0.47 g, 1.50 mmol) in anhydrous methanol (33 ml) and anhydrous CH₂Cl₂(5 ml) was treated with tert-butyl 4-aminobenzoate (0.34 g, 1.78 mmol)followed by decaborane (0.07 g, 0.58 mmol) and the mixture stirred atroom temperature under argon for 18 h. The solvent was removed in vacuoand the residue purified by column chromatography (50 g of silica gel)eluting with 30% ethyl acetate in CH₂Cl₂ to yield the desired product asa white solid (0.43 g, 58%); m.p. 231° C.; ¹H-NMR (CDCl₃) δ 1.26 (s, 9H,CMe₃), 1.58 (s, 9H, CO₂CMe₃), 2.00 (m, 1H, 7-H), 2.72 (m, 1H, 7-H), 3.08(m, 2H, 8-H), 5.10 (s, 2H, 2-CH₂), 5.15 (m, 1H, 6-H), 6.67 (d, J=8.8 Hz,2H, 3′-H, 5′-H), 7.58 (s, 1H, 9-H), 7.87 (d, J=8.8 Hz, 2H, 2′-H, 6′-H),8.24 (s, 1H, 5-H); MS (FAB, m/z). Found 491 [(M+H)⁺, 25%], 514 [(M+Na)⁺,100%]. Found C, 68.37; H, 6.86; N, 8.35. C₂₈H₃₃N₃O₅ requires C, 68.41;H, 6.77; N, 8.55%.

tert-Butyl4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoate

A suspension of (propargyl)Co₂(CO)₆ ⁺BF₄ ⁻ (213 mg, 0.52 mmol) inanhydrous CH₂Cl₂ (25 ml) was treated with tert-butyl4[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-amino]benzoate(200 mg, 0.41 mmol) and the red solution stirred at room temperatureunder argon for 15 minutes. Diisopropylethylamine (0.15 ml, 0.86 mmol)was added and the mixture stirred at room temperature under argon for 1h. The mixture was partitioned between ethyl acetate (30 ml) and brine(30 ml). The organic extract was dried (Na₂SO₄) and the solvent removedin vacuo. The residue was purified by column chromatography (20 g ofsilica gel) eluting with a gradient of 0-10% ethyl acetate in CH₂Cl₂ toyield the complex as a red oil (191 mg, 58%); ¹H-NMR (CDCl₃) δ 1.26 (s,9H, CMe₃), 1.59 (s, 9H, CO₂CMe₃), 2.31 (m, 1H, 7-H), 2.62 (m, 1H, 7-H),3.13 (m, 2H, 8-H), 4.57 (AB system, J=16.9 Hz, 2H, propargyl CH₂), 5.09(s, 2H, 2-CH₂), 5.63 (t, J=8.3, 1H, 6-H), 5.98 (s, 1H, propargyl CH),6.91 (d, J=8.9 Hz, 2H, 3′-H, 5′-H), 7.61 (s, 1H, 9-H), 7.90 (d, J=8.9Hz, 2H, 2′-H, 6′-H), 8.14 (s, 1H, 5-H), 10.25 (br s, 1H).

A solution of this complex (186 mg, 0.23 mmol) in ethanol (30 ml) wastreated with Fe(NO₃)₃.9H₂O (1.1 g) and the solution stirred at roomtemperature for 2 h. The solution was partitioned between ethyl acetate(30 ml) and water (30 ml). The organic extract was washed with brine (30ml), dried (Na₂SO₄) and the solvent removed in vacuo. The residue waspurified by column chromatography (20 g of silica gel) eluting with 10%ethyl acetate in CH₂Cl₂ to yield the desired product as a white solid(94 mg, 78%); m.p. 134° C.; ¹H-NMR (CDCl₃) δ 1.32 (s, 9H, CMe₃), 1.61(s, 9H, CO₂CMe₃), 2.23 (s, 1H, propargyl CH), 2.38 (m, 1H, 7-H), 2.62(m, 1H, 7-H), 3.07 (m, 1H, 8-H), 3.25 (m, 1H, 8-H), 3.94 (AB system,J=18.6 Hz, 2H, propargyl CH₂), 5.12 (s, 2H, 2-CH₂), 5.68 (t, J=8.2 Hz,1H, 6-H), 6.99 (d, J=9.1 Hz, 2H, 3′-H, 5′-H), 7.63 (s, 1H, 9-H), 7.95(d, J=9.0 Hz, 2H, 2′-H, 6′-H), 8.16 (s, 1H, 5-H), 9.55 (br s, 1H); MS(ESI, m/z) 552 {(M+Na)⁺, 100%}, 530 {(M+H)⁺, 20%}. Found C, 70.14; H,6.80; N, 7.73. C₃₁H₃₅N₃O₅ requires C, 70.30; H, 6.66; N, 7.93%.

4-[N-((6RS)-2-(2,2-Dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoicacid

A solution of tert-butyl4-[N-((6R,S)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]-benzoate(80 mg, 0.15 mmol) in trifluoroacetic acid (5 ml) was stirred at roomtemperature with protection from the light for 1.5 h. The solvent wasremoved in vacuo and the residue triturated with 1:1 diethyl ether andhexane to yield the desired product as a white solid (81 mg, TFA salt);m.p. 133° C.; ¹H-NMR (DMSO-d₆) δ1.23 (s, 9H, CO₂CMe₃), 2.22 (m, 1H,7-H), 2.50 (m, 1H, 7-H), 3.03 (m, 2H, 8-H), 3.14 (s, 1H, propargyl CH),3.97 (AB system, J=18.8 Hz, 2H, propargyl CH₂), 4.95 (s, 2H, 2-CH₂),5.79 (t, J=8.6 Hz, 1H, 6-H), 7.03 (d, J=9.0 Hz, 2H, 3′-H, 5′-H), 7.51(s, 1H, 9-H), 7.81 (d, J=6.6 Hz, 2H, 2′-H, 6′-H), 7.83 (s, 1H, 5-H).

Tri-tert-butylN-{N-{4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamate

A solution of4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoicacid (80 mg, 0.15 mmol) in anhydrous dimethylformamide (7 ml) wastreated with tri-tert butyl-L-γ-glutamyl-D-glutamate (150 mg, 0.33mmol), diethyl cyano-phosphonate (0.06 ml, 0.40 mmol) and triethylamine(0.06 ml, 0.40 mmol). The solution was stirred at room temperature underargon with protection from the light for 2.5 h. The solution waspartitioned between ethyl acetate (25 ml) and water (25 ml). The aqueouslayer was extracted with ethyl acetate (2×20 ml). The combined organicextracts were washed with 10% aqueous citric acid (2×30 ml), saturatedaqueous NaHCO₃ (30 ml), dilute brine (30 ml), dried (Na₂SO₄) and thesolvent removed in vacuo. The residue was purified by columnchromatography (30 g of silica gel) eluting with 40% ethyl acetate inCH₂Cl₂ to yield the desired product as a white solid (94 mg, 62%); m.p.109° C.; ¹H-NMR (CDCl₃) δ 1.29 (s, 9H, —COCMe₃), 1.43 (s, 9H, COOCMe₃),1.47 (s, 9H, COOCMe₃), 1.48 (s, 9H, COOCMe₃), 1.60-2.10 (m, 5H, 2×gluβ-CH₂, 7-CH), 2.21 (s, 1H, propargyl CH), 2.22-2.50 (m, 4H, 2×gluγ-CH₂), 2.59 (m, 1H, 7-H), 3.08 (m, 1H, 8-H), 3.20 (m, 1H, 8-H), 3.92(AB system, J=19.0 Hz, 2H, propargyl CH₂), 4.48, 4.76 (2×m, 2H, 2×gluα-CH), 5.12 (s, 2H, 2-CH₂), 5.64 (t, J=8.1 Hz, 1H, 6-H), 6.99 (d, J=8.8Hz, 2H, 3′-H, 5′-H), 7.07 (m, 2H, 2×CONH), 7.64 (s, 1H, 9-H), 7.80 (d,J=8.8 Hz, 2H, 2′-H, 6′-H), 8.13 (s, 1H, 5-H); MS (ESI, m/z) 922{(M+Na)⁺, 100%}, 900 {(M+H)⁺, 40%}. Found C, 64.85; H, 7.23; N, 7.33.C₄₉H₆₅N₅O₁₁.0.5H₂O requires C, 64.76; H, 7.27; N, 7.71%.

N-{N-{4-[N-((6RS)-2-Hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid

Tri-tert-butylN-{N-{4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]-benzoyl}-L-γ-glutamyl}-D-glutamate(80 mg, 0.09 mmol) was dissolved in trifluoro-acetic acid (5 ml) andstirred at room temperature with protection from the light for 1 h. Thesolvent was removed in vacuo and the residue dissolved in methanol (3ml) and water (3 ml). The pH of the solution was adjusted to pH 12 with1M sodium hydroxide solution and stirred at room temperature for 6 h.The solution was acidified to pH 4 with 1M hydrochloric acid and cooledto 0° C. The precipitate was collected by filtration and dried undervacuum over P₂O₅ to yield the desired product as a pale brown solid (27mg, 47%); m.p. 172° C.; ¹H-NMR (DMSO-d₆) δ 1.60-2.10 (m, 5H, 2×gluβ-CH₂, 7-CH), 2.15-2.40 (m, 5H, 2×glu γ-CH₂, 7-H), 2.99 (m, 1H, 8-H),3.12 (s, 1H, propargyl CH), 3.16 (m, 1H, 8-H), 3.98 (AB system, J=19.9Hz, 2H, propargyl CH₂), 4.18, 4.30 (2×m, 2H, 2×glu α-CH), 4.36 (s, 2H,2-CH₂), 5.58 (br 1H, —OH), 5.77 (t, J=7.9 Hz, 1H, 6-H), 7.01 (d, J=8.9Hz, 2H, 3′-H, 5′-H), 7.54 (s, 1H, 9-H), 7.80 (d, J=8.5 Hz, 2H, 2′-H,6′-H), 7.82 (s, 1H, 5-H), 8.15 (d, J=7.5 Hz, 1H), 8.35 (d, J=7.2 Hz, 1H)(2×CONH); MS (ESI, m/z) 670 {(M+Na)⁺, 45%}, 648 {(M+H)⁺, 100%}; HRMS:measured 648.2313; calculated for C₃₂H₃₅N₅O₁₀ (M+H)⁺: 648.2306.

EXAMPLE 3 Synthesis of CB300960 (N-methyl Derivative of CB300945)

4-{N-[(6RS)-2-Hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoicacid

Method A: A solution of tert-butyl4-{N-[(6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoate(0.150 g, 0.28 mmol) in dichloromethane (2 ml) and trifluoroacetic acid(6 ml) was stirred at room temperature for 1 hour. The solvents werethen removed in vacuo, and the residue was suspended in methanol (3 ml)and water (5 ml). The pH was adjusted to ˜10 with 1N NaOH (1.1 ml), andthe mixture was stirred at room temperature for 4 hours. The reactionmixture was diluted with water (5 ml) and the pH was adjusted to ˜5 with1N HCl. The solid was then collected by filtration, but ¹H-NMR indicatedno complete removal of the pivaloyl group. This solid was suspended intothe filtrate and then 1N NaOH (0.9 ml, 0.9 mmol) was added (pH ˜12). Themixture was stirred at room temperature for 3.5 hours, then more 1N NaOH(0.2 ml) was added, and the mixture was stirred at room temperature fora further 0.5 hours. The pH was then adjusted to ˜5.0 with 1N HCl. Theoff-white precipitate was collected by filtration, washed with water,and dried in vacuo over P₂O₅: 0.086 g, (79%); ¹H-NMR (250 MHz, DMSO-d₆,TMS) 2.22 (m, 1H 7-CH), 2.90-3.30 (m, 3H, C≡CH, 8-H), 3.97 (ABq, J=18.6Hz, 2H, CH₂C≡C), 4.37 (d, J=6.1 Hz, 2H, 2-CH₂), 5.56 (t, 1H, CH₂OH),5.78 (t, J=7.51 Hz, 1H, 6-H), 7.03 (d, J=8.9 Hz, 2H, 3′,5′-H), 7.55 (s,1H, 9-H), 7.82 (m, 3H, 2′,6′-H); MS (ESI, m/z) 779 {(2M+H)⁺, 100%}, 390{(M+H)⁺, 60%}.

Method B: A solution of tert-butyl4-{N-[(6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}-benzoate(0.050 g, 0.11 mmol) in dichloromethane (1 ml) and trifluoroacetic acid(2.4 ml) was stirred at room temperature for 1 hour. The solvents werethen removed in vacuo, and the residue was triturated with diethylether. The off-white precipitate was collected by filtration, and washedwith ether to obtain the desired product as the trifluoroacetate salt:0.044 g.

Tri-tert-butylN-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamate

To a mixture of4-{N-[(6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoicacid (0.075 g, ˜0.19 mmol), tri-tert-butylL-γ-glutamyl-N-methyl-L-glutamate (V. Bavetsias et al., J. Med. Chem.,1997, 40, 1495-1510; 0.110 g, 0.24 mmol), and anhydrous DMF (2.0 ml) wasadded diethyl cyanophosphonate (0.036 g, 0.22 mmol) with the aid ofanhydrous DMF (0.2 ml) followed by triethylamine (0.022 g, 0.22 mmol).The clear solution was stirred at room temperature for 1.5 hours, thenit was partitioned between ethyl acetate (50 ml) and brine (40 ml). Theaqueous layer was extracted with more ethyl acetate (2×50 ml). Thecombined organics were washed with 10% aqueous citric acid (40 ml),saturated sodium bicarbonate solution (40 ml), and brine (40 ml), dried(Na₂SO₄), and concentrated in vacuo. Purification by columnchromatography, on elution with a gradient of methanol indichloromethane (0 to 6%), afforded an off-white solid that was furtherpurified by trituration with hexane/dichloromethane/diethyl ether: 0.062g (40%); mp 116-120° C. (softens); ¹H-NMR (250 MHz, DMSO-d₆, TMS) 1.36,1.37, 1.38, 1.41 (4×s, 27H, 3×C(CH₃)₃), 1.70-2.35 (m) and 2.50 (mobscured by DMSO peak) (10H, 2×β-CH₂, 2×γ-CH₂, 7-CH₂), 2.63, 2.82 (2×s,3H, CONMe), 2.90-3.25 (m, 3H, C≡CH, 8-H), 3.97 (ABq, J=17.6 Hz, 2H,CH₂C≡C), 4.32 (m, 1H, glu α-CH), 4.38 (d, J=6.1 Hz, 2H, 2-CH₂), 4.50,4.82 (2×dd, 1H, Meglu α-CH), 5.56 (t, J=6.9 Hz, 1H, CH₂OH), 5.78 (t,J=7.10 Hz, 1H, 6-H), 7.02 (d, J=8.6 Hz, 2H, 3′,5′-H), 7.55 (s, 1H, 9-H),7.78 (d, J=8.9 Hz, 2H, 2′,6′-H), 7.82 (s, 1H, 5-H), 8.32 (m, 1H, CONH),11.81 (s, 1H, N³—H); MS (ESI, m/z) 830 {(M+H)⁺, 100%}.

N-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamicacid

A solution of tri-cert-butylN-{N-{4-[N-((6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]-benzoyl}-L-γ-glutamyl}-N-methyl-L-glutamate(0.060 g, 0.07 mmol) in trifluoroacetic acid (3.5 ml) was stirred atroom temperature for 1 hour and 10 min with protection from the light.The solvent was then removed in vacuo and the residue was suspended inwater (6 ml). The pH was adjusted to ˜10 with 1N NaOH, then to ˜4 with1N hydrochloric acid. The white precipitate was collected by filtration,and dried in vacuo over P₂O₅: 0.035 g (77%), mp>165° C. (dec); ¹H-NMR(250 MHz, DMSO-d₆, TMS) 1.80-2.35 (m) and 2.50 (m obscured by DMSO peak)(10H, 2×β-CH₂, 2×γ-CH₂, 7-CH₂), 2.66, 2.83 (2×s, 3H, CONMe), 2.90-3.25(m, 3H, C≡CH, 8-H), 3.97 (ABq, J=18.4 Hz, 2H, CH₂C≡C), 4.32 (m obscured,1H, glu α-CH), 4.38 (d, J=5.6 Hz, 2H, 2-CH₂), 4.55, 4.91 (2×dd, J=10.0,4.5 Hz, 1H, Meglu α-CH), 5.56 (poorly resolved t, 1H, CH₂OH), 5.77 (t,J=8.06 Hz, 1H, 6-H), 7.02 (d, J=7.8 Hz, 2H, 3′,5-H), 7.55 (s, 1H, 9-H),7.81 (d, J=10.1 Hz, 3H, 2′,6′-H, 5-H), 8.32 (m, 1H, CONH), 11.82 (s, 1H,N³—H); MS (ESI, m/z) 662 {(M+H)⁺, 100%}. Found: C, 57.52; H, 5.52; N,10.17; C₃₃H₃₅N₅O₁₀1.5 H₂O requires: C, 57.55; H, 5.56; N, 10.17%.

EXAMPLE 4 In Vitro Evaluation

CB300945, the (RS)-C2-CH₂OH analogue of the knowncyclopenta[g]quinazoline, CB300638 was synthesised as in Example 1. Inaddition, the C2-NH2 was made as a comparative example. The activity ofthis new series of cyclopenta[g]quinazolines with C2-CH₃ (RS-CB300638),C2-CH₂OH (RS-CB300945) and C2-NH₂ (RS-CB300944) substitutions werecompared with three other series of quinazoline derivatives in mouseL1210-FBP cells and human tumour cell lines co-expressing the RFC andα-FR. The human A431-FBP cell line was transfected with the α-FR andsensitivity compared with the A431 cell line. Human nasopharengeal KBcells constitutively overexpress this receptor (see Bagnoli et al.Oncogene, 19, 4754-4763, 2000).

In the quinazoline series, the C2-CH₂OH analogues were found to havelower affinities for the α-FR of L1210-FBP cells (0.21 to 0.29) than the2-CH₃ (0.37 to 0.54) and particularly the 2-NH₂ (1.3 to 1.7)counterparts. In the cyclopenta[g]quinazoline series the C2-CH₃ analogue(CB300638) had a relative binding affinity of 0.57. However,unexpectedly the C2-CH₂OH analogue (CB300945) had a higher affinity thanpredicted (0.71) and the C2-NH₂ analogue (CB300944) had one lower thanpredicted (0.57). Thus in the cyclopenta[g]quinazoline seriesC2-substitution did not markedly affect binding to the α-FR. A similarpattern of binding was noted for α-FR expressed by A431-FBP cells (datanot shown).

A431-FBP and KB cells were highly sensitive to the C2-CH₃cyclopenta[g]quinazoline with a L-glu-γ-D-glu ligand (S or RS-CB300638).However, very little activity of the C2-NH₂ analogue (RS-CB300944) couldbe attributed to α-FR mediated uptake into either of these cell lines(Tables 13). On the other hand the C2-CH₂OH analogue (RS-CB300945) washighly potent in A431-FBP and KB cells and more highly selective forthese cells compared with A431 cells, than RS-CB300638.

Tables 4 and 5 show the structures of the compounds tested.

EXAMPLE 5 Formulation

The following illustrate representative pharmaceutical dosage formscontaining a cyclopenta[g]quinazoline of formula (I), particularly inpharmaceutically acceptable salt form, for therapeutic or prophylacticuse in humans:

(a) Tablet I mg/tablet Cyclopenta[g]quinazoline salt 100 Lactose Ph.Eur. 182.75 Croscarmellose sodium 12.0 Maize starch paste (5% w/v paste)2.25 Magnesium stearate 3.0 (b) Tablet II mg/tabletCyclopenta[g]quinazoline salt 50 Lactose Ph. Eur. 223.75 Croscarmellosesodium 6.0 Maize starch 15.0 Polyvinylpyrrolidone (5% w/v paste) 2.25Magnesium stearate 3.0 (c) Tablet III mg/tablet Cyclopenta[g]quinazolinesalt 1.0 Lactose Ph. Eur. 93.25 Croscarmellose sodium 4.0 Maize starchpaste (5% w/v paste) 0.75 Magnesium stearate 1.0 (d) Capsule mg/capsuleCyclopenta[g]quinazoline salt 10.0 Lactose Ph. Eur. 488.5 Magnesiumstearate 1.5 (e) Injection I (50 mg/ml) Cyclopenta[g]quinazoline salt5.0% w/v 1M Sodium hydroxide solution 15.0% v/v  0.1M Hydrochloric acid(to adjust pH to 7.6) Polyethylene glycol 400 4.5% w/v Water forinjection to 100% (f) Injection II 10 mg/ml Cyclopenta[g]quinazolinesalt 1.0% w/v Sodium phosphate BP 3.6% w/v 0.1M Sodium hydroxidesolution 15.0% v/v  Water for injection to 100% (1 mg/ml, buffered (g)Injection III to pH 6) Cyclopenta[g]quinazoline salt  0.1% w/v Sodiumphosphate BP 2.26% w/v Citric acid 0.38% w/v Polyethylene glycol 400 3.5% w/v Water for injection to 100%

The above formulations may be prepared by conventional procedures wellknown in the pharmaceutical art. The tablets (a) to (c) may be entericcoated by conventional means, for example with a coating of celluloseacetate phthalate.

TABLE 1 Activity of 2-substituted quinazolines andcyclopenta[g]quinazolines in human A431 and A431-FBP cell lines grown in20 nM folate (RSLV) Inhibition of cell growth, IC₅₀, μM Inhibition ofcell growth, IC₅₀, μM 20 nM LV 20 nM LV A431 + 1 μM FA A431-FBPA431-FBP + 1 μM FA (fold increased IC₅₀ in (fold increased sensitivity(fold increased IC₅₀ in A431 presence of folic acid) compared with A431)presence of folic acid) CB30195 0.4 ± 0.14 0.37 ± 0.14 0.17 ± 0.065 0.23± 0.87  (1)  (1) CB300612 2.2 ± 0.78  1.9 ± 0.65 1.2 ± 0.17 1.3 ± 0.23(1)  (2)  (1) CB300604 3.7 ± 0.71  3.4 ± 0.91 0.28 ± 0.057 1.2 ± 0.59(1)  (13)  (4) ZD9331 0.082 ± 0.042  0.067 ± 0.029 0.018 ± 0.0097 0.034± 0.0087 (1)  (4)  (2) CB300512 — — — — CB300533 — — — — CB300395 0.72 ±0.18  0.72 ± 0.12 0.47 ± 0.20  0.50 ± 0.17  (1)  (2)  (1) CB300635 9.5 ±5.1  9.7 ± 5.4 4.1 ± 2.0  4.3 ± 2.0  (1)  (2)  (1) CB300616 5.9 ± 1.0 6.2 ± 1.6 4.6 ± 0.40 4.7 ± 1.1  (1)  (1)  (1) CB300638 (RS) 1.4 ± 0.23 1.4 ± 0.25 0.0065 ± 0.0001  0.87 ± 0.29  (1) (220) (130) CB300944 (RS)23, 27 22, 25 8.3 ± 0.64 7.1 ± 1.0  (1)  (3)  (1) CB300945 (RS) 9.8 ±3.4  9.3 ± 3.5 0.0021 ± 0.0011  6.5 ± 0.86 (1) (4700)  (3100)  CB300960(R,S) 4.5 4.3 0.028 5.1 (1) (180)

TABLE 2 Activity of 2-substituted quinazolines andcyclopenta[g]quinazolines in human A431 and A431-FBP cell lines grown in1 nM folate (RS LV) Inhibition of cell growth, IC₅₀, μM Inhibition ofcell growth, IC₅₀, μM 1 nM LV 1 nM LV A431 + 1 μM FA A431-FBP A431-FBP +1 μM FA (fold increased IC₅₀ in (fold increased sensitivity (foldincreased IC₅₀ in A431 presence of folic acid) compared with A431)presence of folic acid) CB30195 0.25 ± 0.13 0.21 ± 0.078 0.10 ± 0.0320.22 ± 0.085 (1) (3) (2) CB300612  1.8 ± 0.91 1.5 ± 0.51 1.8 ± 0.68 2.9± 0.55 (1) (1) (1) CB300604  1.6 ± 0.36 2.0 ± 0.49 0.50 ± 0.015  1.8 ±0.058 (1) (3) (4) ZD9331 0.061 ± 0.014 0.056 ± 0.025  0.0088 ± 0.0052 0.027 ± 0.0076 (1) (7) (3) CB300512 — — — — CB300533 — — — — CB300395 0.70 ± 0.072 0.74 ± 0.053 0.43 ± 0.36  0.54 ± 0.34  (1) (2) (1)CB300635 6.1 ± 1.3 6.7 ± 1.9  2.7 ± 0.87 3.3 ± 1.5  (1) (2) (1) CB3006164.9 ± 2.0 5.2 ± 2.1  5.5 ± 3.5  5.6 ± 3.0  (1) (1) (1) CB300638 (RS) 1.2 ± 0.21 0.98 ± 0.23  0.0072 ± 0.001  0.61 ± 0.012 (1) (170)  (85) CB300944 (RS)  24 ± 3.6 24 ± 2.9  9.1 ± 0.83 12 ± 2.1  (1) (3) (1)CB300945 (RS) 7.2 ± 3.6 7.3 ± 3.3  0.0019 ± 0.0006  5.7 ± 1.3  (3800)  (3100)  

TABLE 3 Activity of 2-substituted quinazolines andcyclopenta[g]quinazolines in human KB cells grown in 1 and 20 nM folate(RS LV) Inhibition of cell growth, IC₅₀, μM Inhibition of cell growth,IC₅₀, μM 1 nM LV 20 nM LV KB + 1 μM FA KB + 1 μM FA (fold increased IC₅₀in (fold increased IC₅₀ in KB presence of folic acid) KB presence offolic acid) CB30195 0.0053 ± 0.0006 0.18 ± 0.012 0.027 ± 0.002 0.15 ±0.015  (34)  (6) CB300612 0.97 ± 0.20 2.2 ± 0.35  1.7 ± 0.20 2.0 ± 0.25 (2)  (1) CB300604  0.0061 ± 0.00095 0.69 ± 0.08  0.035 ± 0.005 0.65 ±0.04  (120)  (20) ZD9331 0.0021 ± 0.0013 0.00641 ± 0.0035  0.0036 ±0.0021 0.012 ± 0.005   (3)  (3) CB300512 — — — — CB300533 — — — —CB300395 0.063 ± 0.081 0.23 ± 0.05   0.065 ± 0.0092 0.17 ± 0.032  (4) (3) CB300635 2.8 ± 1.1 3.8 ± 0.6  3.2 ± 0.6 4.4 ± 0.25  (1)  (1)CB300616 0.0037 ± 0.0018 0.58 ± 0.05   0.008 ± 0.00072 0.59 ± 0.19 (160)  (74) CB300638 (RS) 0.0079 ± 0.001  0.61 ± 0.012 0.0053 ± 0.00250.76 ± 0.17   (76) (140) CB300944 (RS) 19 ± 0  19 ± 1.5    19 ± 0.58 19± 1.2   (1)  (1) CB300945 (RS) 0.0034 ± 0.0007 7.6 ± 1.3  0.0034 ±0.0009 7.3 ± 2.0  (2200)  (2100) 

TABLE 4 Structures of the compounds tested-Comparative compounds

ZD9331, R = Me CB300512, R = NH₂ CB300533, R = CH₂OH

CB300395, R = Me CB300635, R = NH₂ CB300616, R = CH₂OH

CB30195, R = Me CB300612, R = NH₂ CB300604, R = CH₂OH

TABLE 5 Structures of the compounds tested-Compounds of the invention

CB300945

CB300638

CB300944

CB300960

The invention claimed is:
 1. A method for aiding regression andpalliation of a cancer which over expresses the alpha isoform of thefolate receptor relative to normal tissue in a patient in need of suchtreatment which comprises administering to said patient an effectiveamount of a cyclopenta[g]quinazoline of the formula I

wherein: A is a group OR⁰ or NR⁰R¹ wherein R⁰ and R¹ are eachindependently hydrogen or C₁₋₄ alkyl; p is an integer in the range 1 to4; R² is ethyl, propyl, prop-2-enyl, prop-2-ynyl, 2-hydroxyethyl,2-fluoroethyl, 2-bromoethyl or 2-cyanoethyl; Ar¹ is 1,4-phenylene whichmay optionally bear 1 or 2 substituents selected from the groupconsisting of chloro and fluoro: or Ar¹ is thiophen-2,5-diyl, thiazol2-2,5-diyl or pyridine-2,5-diyl; and R³ is a group of the formula:-A⁵-CON(R)CH(Y⁴)Y⁵ in which A⁵ is a C₁₋₆ alkylene group and R ishydrogen, C₁₋₄ alkyl, C₃₋₄ alkenyl or C₃₋₄ alkynyl; Y⁴ is carboxy,tetrazol-5-yl, N—(C₁₋₄ alkylsulfonyl)carbamoyl,N-(phenylsulfonyl)carbamoyl which may optionally bear one or twosubstituents on the phenyl ring selected from the group consisting ofhalogeno, nitro, C₁₋₄ alkyl and C₁₋₄ alkoxy, tetrazol-5-ylthio,tetrazol-5-ylsulfinyl or tetrazol-5-ylsulfonyl; and Y⁵ is the residue ofa naturally occurring amino acid selected from the group consisting of:—CH₃, —(CH₂)₃NHC(NH₂)═NH, —CH₂CO₂H, —CH₂SH, —CH(CH₃)CH₂CH₃,—CH₂CH(CH₃)CH₃, —(CH₂)₃NH₂, —CH₂C₆H₅, —CH₂OH, —CH(CH₃)₂ and —CH₂CH₂CO₂H;the compound (I) optionally being in the form of a pharmaceuticallyacceptable salt or ester.
 2. A method according to claim 1 wherein: A isa group OR⁰ in which R⁰ is hydrogen or methyl; R² is ethyl orprop-2-ynyl; and Ar¹ is 1,4-phenylene or 1,4-phenylene having a2-fluoro.
 3. A method according to claim 1 in which R³ is a group suchthat NH₂CH(COOH)R³ is N-L-γ-glutamyl-D-glutamic acid.
 4. A methodaccording to claim 1 wherein p is
 1. 5. A method for aiding regressionand palliation of a cancer which over expresses the alpha isoform of thefolate receptor relative to normal tissue in a patient in need of suchtreatment which comprises administering to said patient an effectiveamount of a cyclopenta[g]quinazoline selected fromN-{N-{4-[N-((6S)-2-methoxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid; andN-{N-{4-[N-((6S)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamicacid; or a pharmaceutically acceptable salt or ester thereof.
 6. Amethod according to claim 1 or claim 5 wherein the cancer comprises acarcinoma of ovarian origin.
 7. A method according to claim 1 wherein Y⁵is —CH₂CH₂CO₂H.
 8. A method according to claim 1 wherein Y⁴ is carboxyor tetrazol-5-yl.
 9. A method according to claim 1 wherein the alphacarbon atom of the group —CONHCH(CO₂H)— has the L configuration.
 10. Amethod according to claim 1 or claim 5 comprising administering, to saidpatient an effective amount of a cyclopenta[g]quinazoline according toclaim 1 or claim 5 and one or more additional anti cancer substances.11. A method according to claim 1 or claim 5 comprising administering,to said patient an effective amount of a cyclopenta[g]quinazolineaccording to claim 1 or claim 5 and one or more additional anti cancersubstances selected from other anti metabolites, DNA interacting agents,signal transduction inhibitors and other inhibitors of dysregulatedpathways in tumours.